Epoxide hydrolases of aspergillus origin

ABSTRACT

The invention concerns proteins of fungal origin having an epoxide hydrolase activity, such as those obtained in essentially pure form by extraction from fungi cells, or by culturing in host cells transformed by a nucleotide sequence coding for said fungal proteins. The invention also concerns the uses thereof, in particular for implementing methods for preparing enantiopure epoxides and/or diols.

[0001] The present invention relates to proteins of fungal origin orproteins derived from the latter, possessing epoxide hydrolase activity,as well as their uses, notably for the preparation of enantiomericallypure (or enantiopure) molecules, such as epoxides and/or vicinal diolsof high enantiomeric purity.

[0002] The epoxides or the vicinal diols are important compounds inorganic synthesis. If they have a chiral structure, they can be usedeither in the racemic form, or in the optically enriched or evenenantiomerically pure form. In the first case they constitute baseproducts for the chemical industry (polymerizable monomers or componentsof industrial products such as glycol, propyleneglycol etc.). In thesecond case they can be used as chiral synthons for the production ofvarious optically pure products, for example biologically activemolecules marketed by the pharmaceutical or plant-protection industry,or materials with specific optical properties (e.g. liquid crystals).

[0003] This is why various strategies of chemical synthesis have beenelaborated for carrying out their production. With regard to theproduction of diols, these strategies often involve the hydrolysis of anepoxide in a more or less concentrated acidic or basic inorganic medium,which in itself gives rise to additional costs due to reprocessing ofthe mother liquors and/or the salts formed in the course of the process.

[0004] When these molecules have to be produced in an optically activeform, several strategies have been described and developed (Schurig etal. 1992, Pedragosa-Moreau et al. 1995). For example, theKatzuki-Sharpless oxidation reaction makes it possible to convert anolefin into an optically enriched epoxide by means of a titanium-basedchiral organometallic catalyst. However, this approach is limited toolefins bearing an alcohol group in the alpha position of the doublebond, necessary for coordination of the catalyst.

[0005] Other methods have been developed more recently, and they too arebased for the most part on the use of organometallic catalysts veryoften involving heavy metals such as manganese or cobalt. However,although they are very effective on certain types of substrates, theyonly display average selectivities on other families of molecules. Inall cases, they are difficult to use in industrial conditions, owing tothe technical constraints imposed by the use of the heavy metalsinvolved.

[0006] Various techniques of biocatalysis have been described forovercoming this problem. They employ indirect strategies involving theuse of enzymes such as lipases, peroxidases or monooxygenases (Archelaset al. 1997). However, most of these approaches require the developmentof expensive systems for the recycling of cofactors, once again makingthem particularly difficult and expensive to implement in preparativeconditions.

[0007] The use of an enzyme that makes it possible to effect directhydrolysis of an epoxide therefore represents an interesting andoriginal means of direct preparation of optically enriched epoxides orof achiral, racemic or optically enriched diols. These enzymes, calledepoxide hydrolases, offer the advantage on the one hand of not requiringa cofactor and, on the other hand, of making it possible to effect theaddition of a water molecule to an epoxide in particularly mildconditions. If the substrate is chiral, and depending on theenantioselectivity and the regioselectivity of this addition process,the diol obtained will be racemic or enantiomerically enriched (Archelaset al. 1998).

[0008] Although numerous works have been devoted to this type of enzymepresent in mammals, their use in organic synthesis cannot be envisagedowing to the difficulty of obtaining them in sufficient quantity.

[0009] The possibility of using epoxide hydrolases of microbial origin(bacteria, yeasts, fungi)—which can be produced in large quantities bysimple microbial fermentation—as a “tool” in organic synthesis wouldtherefore constitute a considerable advance.

[0010] Examples of preparation of optically active compounds by usingmicroorganisms as biocatalysts have been described, but only relate touncharacterized enzymatic activities detected in various microorganisms.Thus, in European patent application EP 611 826 (Daicel ChemicalIndustries Co. Ltd.), the examples of microorganisms given, capable ofproducing an (S) optically active epoxide starting from a racemicepoxide, are in particular a strain of microorganism belonging to thegenus Candida, Rhodosporidium, Rhodococcus and Nosardioides. Theexamples of microorganisms capable of producing an (R) optically activeepoxide are, in particular, a strain of microorganism belonging to thegenus Thichosporon, Geotrichum, Corynebacterium, Micrococcus, andBrevibacterium.

[0011] Styrene oxide is one of the test substrates finding widestapplication in studies conducted on mammalian epoxide hydtolases, andvarious derivatives substituted on the aromatic ring of this modelsubstrate have also been investigated in this context (Dansette et al.1978; Westkaemper et al. 1981). More recently, studies carried out withenzymatic activities of microbial origin have also used this modelsubstrate, and the inventors themselves have shown that these moleculescan be hydrolysed enantioselectively by a strain of the fungusAspergillus niger, registered at the Natural History Museum (Paris)under No. LCP521 (Lab. de Cryptogamie, 12 rue Buffon, 75005 Paris,France) (Pedragosa-Moreau et al. 1996).

[0012] Nevertheless, experiments of enantioselective hydrolysis effectedwith the aid of fungi, such as the fungus Aspergillus niger, describedup till now, make use of whole cells or of cellular extracts of thefungus, which creates a number of technical problems in application,does not give good yields, and does not allow the structure of thebiological catalyst employed to be defined.

[0013] The use of well identified and characterized epoxide hydrolasesof fungal origin would make it possible to remedy these drawbacks, butso far it has not been possible to isolate and purify an epoxidehydrolase of fungal origin, suggesting the possibility that such anenzyme would not be sufficiently stable to be completely isolated fromits natural environment.

[0014] The present invention results from the demonstration, by theinventors, of the fact that it is possible to isolate and purify anepoxide hydrolase from fungi. Thus, the present invention follows fromthe identification (by purification, sequencing, cloning) of the enzymeresponsible for the epoxide hydrolase activity of fungi, such as thoseof the Aspergillus species.

[0015] One of the aims of the present invention is to supply novelenzymes with epoxide hydrolase activity of fungal origin.

[0016] Another aim of the present invention is to supply the nucleotidesequences encoding these enzymes.

[0017] A further aim of the invention is to supply host cellstransformed by the aforementioned nucleotide sequences, in which thesaid enzymes are advantageously overexpressed.

[0018] The invention also has the aim of supplying methods of obtainingthe said enzymes, notably by extraction and purification from cells offungi, or by culturing host cells as described above.

[0019] Another aim of the present invention is to supply novel methodsof biocatalysis using the aforementioned enzymes or host cells that areproducers of the said enzymes described above, for the synthesis ofvarious epoxides and/or diols, and these methods give higher yields thanthe methods using whole cells, or cellular extracts of fungi previouslydescribed.

[0020] Accordingly, the invention aims more particularly to supplymethods of hydrolysis of achiral or chiral epoxides offering theadvantage that they can be carried out in particularly mild experimentalconditions, i.e. without employing an organic or inorganic, acidic orbasic reagent, notably in a buffered or unbuffered aqueous medium and/orin the presence of water-miscible or water-immiscible organic solvents.Depending on the intrinsic stereochemical properties of the startingepoxide, these methods result in the production of an achiral, racemicor optically enriched diol or—if the starting epoxide is chiral—in theproduction of one of its two enantiomers in optically enriched form, oreven enantiomerically pure form.

[0021] The invention relates to any protein of fungal origin havingepoxide hydrolase activity, such as is obtained in essentially pure formby extraction from cells of fungi, or by culture of host cellstransformed by a nucleotide sequence coding for the aforementionedfungal protein, or to any protein derived by substitution, suppressionor addition of one or more amino acids of the aforementioned protein offungal origin and possessing epoxide hydrolase activity.

[0022] The aforementioned epoxide hydrolase activity can be measuredusing para-nitrostyrene oxide (pNSO) as substrate, and measuring thequantity of diol formed, notably according to the following method:

[0023] Add 50 μL of the preparation containing the enzyme to 410 μL of0.1 M sodium phosphate buffer pH 7.0 (buffer B) and pre-incubate themixture at 35° C. for 2 min. Then add 40 μL of a 50 mM solution ofracemic pNSO in DMF (final pNSO concentration: 4 mM).

[0024] After 10 min of incubation, stop the reaction by adding 1 mL ofdichloromethane. Stir the mixture vigorously so as to extract both thesubstrate and the diol produced. The quantity of diol formed isdetermined after separation on a column of silica by HPLC (high-pressureliquid chromatography) (Waters Associates, USA) as described previously(Nellaiah et al. 1996).

[0025] One unit of epoxide hydrolase represents the quantity of enzymethat catalyses the formation of one μmol of diol per minute in the aboveconditions. After incubation with raw extracts, the quantity of diolformed increases linearly with time for at least 30 min, and thereaction rate is proportional to the concentration of the enzyme in therange of 0.01 to 1.2 units (Nellaiah et al., 1996).

[0026] The invention relates more particularly to any protein asdescribed above, characterized in that it comprises:

[0027] the sequence SEQ ID NO: 2.

[0028] or any sequence derived from the sequence SEQ ID NO: 2, notablyby substitution, suppression or addition of one or more amino acids, andpossessing epoxide hydrolase activity, the said derived sequencepreferably having a homology of at least 40%, and especially aboveapprox. 80%, with the sequence SEQ ID NO: 2,

[0029] or any fragment of the sequence SEQ ID NO: 2, or a sequencederived from the latter as defined above, and possessing epoxidehydrolase activity, the said fragment preferably consisting of at leastabout 10 amino acids that are contiguous in the region delimited by theamino acids situated at positions 1 and 339 of the sequence SEQ ID NO:2.

[0030] The invention relates more particularly to any protein describedabove, characterized in that it corresponds to a fungal epoxidehydrolase in essentially pure form, such as is obtained by extractionand purification from cultures of cells of fungi of the Aspergillusspecies.

[0031] Accordingly, the invention relates more particularly to anyaforementioned protein, characterized in that it corresponds to thefungal epoxide hydrolase in essentially pure form represented by SEQ IDNO: 2, such as is obtained by extraction and purification from culturesof cells of strains of Aspergillus niger or of Aspergillus turingensis.

[0032] The invention also relates to any protein as described above,characterized in that it corresponds to a recombinant fungal epoxidehydrolase such as is obtained in essentially pure form by transformationof suitable host cells by means of vectors containing:

[0033] the nucleotide sequence SEQ ID NO: 1 encoding the epoxidehydrolase represented by SEQ ID NO: 2, or any sequence derived from SEQID NO: 1 by degeneration of the genetic code, and encoding the epoxidehydrolase represented by SEQ ID NO: 2,

[0034] or any sequence derived from the sequence SEQ ID NO: 1, inparticular by substitution, suppression or addition of one or morenucleotides, and coding for an enzyme possessing epoxide hydrolaseactivity, the said derived sequence preferably having a homology of atleast about 45%, and especially above about 80%, with the sequence SEQID NO: 1,

[0035] or any fragment of the sequence SEQ ID NO: 1, or of a sequencederived from the latter as defined above, and coding for an enzymepossessing epoxide hydrolase activity, the said fragment preferablyconsisting of at least about 20 nucleotides that are contiguous in theregion delimited by the nucleotides situated at positions 1 and 1197 ofthe sequence SEQ ID NO: 1.

[0036] Accordingly, the invention relates more particularly to therecombinant fungal epoxide hydrolase represented by SEQ ID NO: 2, suchas is obtained by transformation of suitable host cells by means ofvectors containing the nucleotide sequence SEQ ID NO: 1, or any sequencederived from SEQ ID NO: 1 by degeneration of the genetic code, andencoding the epoxide hydrolase represented by SEQ ID NO: 2.

[0037] The invention also relates to any nucleotide sequence encoding aprotein of fungal origin with epoxide hydrolase activity as definedabove.

[0038] The invention relates more particularly to any aforementionednucleotide sequence, characterized in that it comprises:

[0039] the sequence represented by SEQ ID NO: 1 encoding the epoxidehydrolase represented by SEQ ID NO: 2,

[0040] or any sequence derived from the sequence SEQ ID NO: 1 bydegeneration of the genetic code, and encoding the epoxide hydrolaserepresented by SEQ ID NO: 2,

[0041] or any sequence derived from the sequence SEQ ID NO: 1,especially by substitution, suppression or addition of one or morenucleotides, and encoding an enzyme possessing epoxide hydrolaseactivity, the said derived sequence preferably having a homology of atleast about 45%, and especially above about 80%, with the sequence SEQID NO: 1,

[0042] or any fragment of the sequence SEQ ID NO: 1, or of a sequencederived from the latter as defined above, and encoding an enzymepossessing epoxide hydrolase activity, the said fragment preferablybeing constituted of at least about 20 nucleotides that are contiguousin the region delimited by the nucleotides situated at positions 1 and1197 of the sequence SEQ ID NO: 1,

[0043] or any complementary nucleotide sequence of the aforementionedsequences or fragments,

[0044] or any nucleotide sequence encoding an enzyme possessing epoxidehydrolase activity, and capable of hybridization with one of theaforementioned sequences or fragments,

[0045] the aforementioned sequences or fragments being in thesingle-stranded or double-stranded form.

[0046] The invention also relates to any vector, especially plasmid,containing a nucleotide sequence as defined above.

[0047] Advantageously, the nucleotide sequences of the invention in theaforementioned vectors, are put under the control of elements thatregulate the expression of the proteins with epoxide hydrolase activitydefined above, notably a promoter, inducible if necessary, and atranscription terminator.

[0048] Preferably, the aforementioned promoter is selected from thosethat permit overexpression of the said proteins in the host cellstransformed by means of the vectors, the said host cells themselvesbeing selected from those that are able to overexpress the saidproteins, especially among the bacteria, viruses, yeasts, fungi, plantsor mammalian cells.

[0049] The invention also relates to any host cell, selected inparticular from bacteria, viruses, yeasts, fungi, plants or mammaliancells, the said host cell being transformed, notably by means of avector as defined above, in such a way that its genome contains anucleotide sequence as mentioned above encoding a protein with epoxidehydrolase activity.

[0050] The invention also relates to the use of proteins with epoxidehydrolase activity as defined above, as enzymatic biocatalysts in theimplementation of methods of preparation of epoxides or ofenantiomerically pure vicinal diols, especially in the pharmaceuticaland plant-protection field, or in the manufacture of specific opticalmaterials.

[0051] Accordingly, the invention relates more particularly to a methodof preparation of epoxides and/or of enantiomerically pure diolsrespectively of the following formulae (II) and (III)

[0052] in which R₁, R₂, R₃ and R₄ represent any groups, and especiallygroups that are characteristic of pharmaceutical and plant-protectioncompounds, or of specific optical materials corresponding to the saidepoxides or vicinal diols,

[0053] the said method comprising a stage of treatment of a mixture ofdiastereoisomeric epoxides, or of a chiral epoxide in racemic form, orof a prochiral epoxide of the following formula (I):

[0054] with a protein with epoxide hydrolase activity as defined above,or with host cells as mentioned above, expressing or overexpressing aprotein with epoxide hydrolase activity as defined above, which leads tothe production of:

[0055] a mixture of the aforementioned compounds of formulae (II) and(III), it being possible, if necessary, for the said compounds offormula (II) and (III) to be separated by an additional stage ofpurification,

[0056] or just the compound of the aforementioned formula (III).

[0057] In the case of production of just the compound of theaforementioned formula (III), this can be effected by a treatment thataccompanies or is subsequent to the treatment described above, notablywith another chemical or enzymatic reagent depending on the startingepoxide, for example with sulphuric acid, especially in the case ofpara-nitrostyrene oxide (Pedragosa-Moreau et al., 1997), or with cellsof the fungus Beauveria sulfurescens, especially in the case of styreneoxide (Pedragosa-Moreau et al., 1993).

[0058] Advantageously, when the method as described above according tothe invention is carried out by means of a protein with epoxidehydrolase activity as defined above, the latter can be immobilized on asolid support such as DEAE cellulose or DEAE Sepharose, or any othersupport or technique that makes it possible to immobilize this enzyme.

[0059] The invention also relates more particularly to the use of aprotein with epoxide hydrolase activity as defined above, in variousforms, including transformed host cells as described above, or of wholecells of fungi, such as Aspergillus niger, producing this enzyme, orsoluble or freeze-dried enzymatic extracts of the said cells, or theenzyme immobilized on a solid support as defined above, in theimplementation of a method as described above for hydrolysis of anachiral epoxide.

[0060] The invention also relates to a method of preparation of proteinwith recombinant epoxide hydrolase activity as defined above,characterized in that it comprises a stage of transformation of hostcells, preferably selected from bacteria, viruses, yeasts, fungi, plantsor mammalian cells, with a vector as described above, and a stage ofpurification of the recombinant epoxide hydrolase produced by the saidcells.

[0061] The invention also relates to a method of preparation of a fungalepoxide hydrolase in essentially pure form, the said method comprising:

[0062] a stage of extraction of the enzyme from cell cultures of fungi,such as fungi of the Aspergillus species, notably by crushing the fungususing a French press or any other suitable means, followed by a stage oflow-speed centrifugation (approx. 10 000 g), recovery of thesupernatant, and concentration by ultrafiltration.

[0063] a stage of purification of the enzyme from the extract obtainedin the preceding stage, notably by successive passages through columnsof DEAE-Sepharose, Phenyl-Sepharose, Mono Q and Superose 12.

[0064] The invention will be further illustrated by the followingdescription of the purification of the epoxide hydrolase from a strainof the fungus Aspergillus niger, as well as cloning of the gene encodingthis epoxide hydrolase, and examples of application of a methodaccording to the invention.

A) Purification and Characterization of an Epoxide Hydrolase fromAspergillus niger with High Enantioselectivity

[0065] 1) Equipment and Methods

[0066] 1) Reagents

[0067] The test substrate used is racemic p-nitrostyrene oxide (pNSO).It is synthesized from ω-bromo-4-nitro acetophenone according to atechnique described by Westkaemper and Hanzlik, 1980. Its pure (R) and(S) enantiomers are obtained from this racemic substrate by a stage ofbiotransformation (Pedragosa-Moreau et al., 1996). Diethylaminoethyl(DEAE)-Sepharose, phenyl-Sepharose, the “Mono Q” and Superose 12 columnsare obtained from Pharmacia LKB (Uppsala, Sweden). H₂[¹⁸O] is obtainedfrom Isotec (Miamisburg, USA) and its [¹⁸O] content is 95%. All theprotein chromatography is effected using the FPLC Pharmacia system at 4°C.

[0068] 2) Organisms, Conditions of Growth and Preparation of Extracts

[0069] The strain of the fungus Aspergillus niger used in this study isregistered at the Natural History Museum (Paris) under No. LCP521 (Lab.de Cryptogamie, 12 rue Buffon, 75005 Paris, France). Culture is carriedout in a fermenter with a capacity of 5 L (liquid volume) in theconditions described by Nellaiah et al., 1996. The cells are harvestedafter 40 h of culture by filtration. They are suspended in 10 mMTris-HCl buffer of pH 7.1 (buffer A) containing 1 mM cysteine, 1 mM EDTAand 0.3 mM phenylmethane sulphonyl chloride (PMSF). An acellular extractis prepared by crushing the fungus using a French press, or any othermeans that can be used by a person skilled in the art, and low-speedcentrifugation (1000 g) in the conditions described by Nellaiah et al.,1996. This extract is concentrated at 100 mL by tangential-flowfiltration using a membrane with a cutoff threshold of 10, 40 or up to100 kDa. Any other manipulation is carried out at a temperature of 4° C.in a buffer solution containing 1 mM cysteine, 1 mM EDTA and 0.3 mM PMSFto avoid inactivating the enzyme. The concentration of the protein isdetermined by the method of Lowry et al., 1951, using bovine serumalbumin as reference.

[0070] 3) Purification of the Epoxide Hydrolase

[0071] The concentrated solution containing the enzyme is deposited on acolumn of DEAE (diethylaminoethyl)-Sepharose (2.5 cm×30 cm) previouslyequilibrated with buffer A containing 0.13 M KCl. The column is washedwith 360 mL of equilibrating buffer, and elution is carried out with alinear gradient of 0.13-0.23 M KCl in buffer A (total volume: 510 mL,flow rate: 3 mL/min, volumes of the fractions: 6 mL).

[0072] The activity is eluted for a potassium chloride concentration of0.17-0.20 M. The active fractions are combined and concentrated to 5 mLby ultrafiltration. The concentrate is deposited on a column ofphenyl-Sepharose (1 cm×10 cm), previously equilibrated with buffer Acontaining (NH₄)₂SO₄ 0.25 M and 21% (v/v) of ethyleneglycol. The columnis washed with 10 mL of the same buffer, and elution is carried out witha linear gradient of ethyleneglycol of 21-56% (v/v) in buffer Acontaining (NH₄)₂SO₄ 0.25 M (total volume: 95 mL, flow rate: 0.5 mL/min,volumes of the fractions: 1 mL).

[0073] The activity is eluted with an ethyleneglycol concentration of30-43% (v/v). The active fractions are combined and concentrated to 5mL. The concentrate is deposited on a Mono Q column (0.5 cm×5 cm),previously equilibrated with Tris-HCl buffer 10 mM, pH 6.5, containing0.13 M KCl. The column is washed with 5 mL of buffer, and elution iscarried out with a linear gradient of potassium chloride of 0.13-0.25 M(total volume 85 mL; flow rate 0.5 mL/min; volumes of the fractions: 1mL). The activity is eluted to a concentration of 0.15-0.16 M ofpotassium chloride. The active fractions are combined and concentratedto 1 mL. The solution containing the enzyme (200 mL) is deposited on acolumn of Superose 12 (1 cm×30 cm) and equilibrated with buffer A (flowrate 0.3 mL/min; volumes of the fractions: 0.6 mL). This stage iscarried out 5 times (200 μL each time) and all the active fractions arecombined. The preparation thus obtained is stored at 4° C.

[0074] 4) Enzymatic Study

[0075] The incubations with H₂[¹⁸O] are carried out in 1 mL flaskscontaining 180 μL of H₂[¹⁸O] buffer B, 20 μL of the purified epoxidehydrolase and 20 μL of substrate (50 mM in acetonitrile). After 1.5 h ofincubation at 25° C. with magnetic stirring (500 rpm) the substrate thatremains and the product formed are extracted with 2 mL ofdichloromethane. The diol is purified by analytical chromatography onsilica (eluent: diethylether). 2 μL of samples are analysed by gaschromatography/mass spectrometry (GC/MS). The diol that remains isconverted to the corresponding acetonide by reaction with2,2-dimethylpropane in the presence of p-toluene sulphonic acid. Theacetonide is analysed by GC/MS as already described by Audier et al.,1968.

[0076] Reaction in a total volume of 5 mL is carried out at 25° C., witha substrate with a concentration of 4.3 mM with DMSO (20 vol.%) asco-solvent in a 0.1 M sodium phosphate buffer pH 8.0.

[0077] The reaction is started by adding 13 U/L of the purified enzyme.Samples are taken every 30 minutes for quantification of theconcentrations of the substrate and of product by HPLC using areversed-phase column (Nellaiah et al., 1996) and for quantification ofthe enantiomeric excess of the epoxide and of the diol by gas-phasechromatography (Nellaiah et al., 1996).

[0078] 5) Polyacrylamide Gel Electrophoresis

[0079] SDS-PAGE electrophoresis is carried out on a plate with thicknessof 1 mm containing a resolving gel (10% of acrylamide) and aconcentrating gel (4% of acrylamide) at pH 8.8 in the presence of 0.1%of sodium dodecyl sulphate (SDS) (Laemmli, 1970). The samples aredissolved in a Tris-HCl buffer (62.5 mM, pH 8.8) containing 1% (w/v) ofSDS, 10% (v/v) of glycerol and 2% (v/v) of β-mercaptoethanol, and areheated at 100° C. for 2 minutes. The proteins are stained with 0.1%(w/v) of Coomassie blue. Migrations on non-denaturing PAGE gels wereeffected in the same way except that no β-mercaptoethanol was added tothe resolving buffer, and that the samples were not heated.Electrofocusing was carried out with a pH gradient of 3-9 by means ofthe Pharmacia LKB “Phastsystem” system and standard Pharmaciaprocedures. The proteins were stained with silver nitrate.

[0080] 6) Determination of Molecular Weight

[0081] The molecular weight was estimated after SDS-PAGE by comparingthe mobility (Rf) of the purified epoxide hydrolase (EH) with that ofthe following reference proteins: phosphorylase B (97.4 kDa), bovineserum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31kDa), trypsin inhibitor (21.5 kDa) and lysozyme (14.4 kDa). Themolecular weight of the native enzyme was estimated from the elutionprofile of Superose 12 by comparing the Kav of the purified EH with thatof the following standard proteins: alcohol dehydrogenase (150 kDa),bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A(25 kDa) and ribonuclease A (13.7 kDa). The exclusion volume and thedead volume were determined using dextran blue and vitamin B12.

[0082] 7) Amino Acid Sequence

[0083] For the amino acid analyses and the N-terminal sequencedeterminations, the peptides were transferred from the SDS gels onto a“glassy-bond” membrane (Biometra, Germany) using standard Bioradprocedures (Hercules, USA). The amino acid composition of the enzyme wasdetermined after acid hydrolysis (6 N HCl at 100° C. under vacuum for 24h) using an automatic amino acid analyser (Beckman 6300 system,Germany). The molecular weight was estimated from the amino acidcomposition using Delaage's method (1968).

[0084] 8) Peptide Sequences

[0085] The proteins were dissolved in an SDS buffer and separated bySDS-PAGE. Part of the gel was stained with Coomassie Blue, and the stripof interest was separated from the rest of the gel. The strip was washedfor 1 h with H₂O, H₂O—CH₃OH (90:10), H₂O—CH₃CN (80:20), and H₂O—CH₃CN(50:50). The strip of gel was then cut into small pieces and dried undervacuum in a Speed-Vac (Savant). Next, 400 μL of a solution containing 25mM Tris-HCl (pH 8.5), 1 mM EDTA, 0.05% SDS, and 5 μg of the proteaseLys-c (Boehringer Mannheim) was added, and the mixture was incubatedovernight at 37° C. The hydrolysate was injected into a reversed-phaseHPLC column (Vydac C₁₈; 2.1×250 mm). The column was eluted at a flowrate of 0.2 mL/min with a linear gradient of 0 to 35% of solution B(CH₃CN, containing 0.07% of trifluoroacetic acid) for 150 min (solutionA consists of water and 0.07% of trifluoroacetic acid) and the peakswere collected and sequenced directly with an Applied Biosystems model477A microsequencer.

[0086] 9) PCR Reaction, Cloning and Sequencing

[0087] The PCR reactions were carried out using degenerated oligomersobtained from partial amino acid sequences as primer and using thegenomic DNA of Aspergillus niger as support. The genomic DNA wasextracted from 1.5 g of mycelium washed with water, ground in liquidnitrogen and suspended in buffer Tris-HCl 50 mM pH 7.5, EDTA 50 mM, SDS3%, β-mercaptoethanol 1%. After reaction for 1 h at 65° C., the solutionwas extracted with a phenol/chloroform/isoamyl alcohol mixture (24/24/1,v/v/v) and chloroform/isoamyl alcohol mixture (24/1), precipitated withisopropanol, and the sediment was dissolved in TE buffer (Tris-HCl 10 mMpH 7.5 and EDTA 1 mM). RNase was added (30 μg/mL) in the course of 1 hat 37° C., the DNA was precipitated with isopropanol, washed in 70%ethanol and dissolved in water. The PCR reactions were carried out in atotal volume of 50 μL, using 100 ng of DNA, dNTP 200 μM, each primer at2 μM and 2 units of Taq polymerase (Perkin Elmer). The PCR reactionswere effected by heating at 95° C. for 5 min, then carried out for 30cycles of amplification at three temperatures (1 min at 95° C., 1 min at58° C., and 1 min at 72° C.). The amplified fragments were cloned at theECOR V site of pBluescript II SK(−) (Statagene) after treatment with oneunit/μL of terminal transferase (Boehringer). The fragments weresequenced using a Pharmacia T7 sequencing kit.

[0088] II) Results

[0089] The EH from Aspergillus niger was purified to electrophoretichomogeneity using a 4-stage chromatographic procedure. In total, 120 μgof the purified enzyme was prepared starting from 24 g of dry mycelium,i.e. from 5 L of culture medium. These relatively low values are due to2 reasons:

[0090] 1) the overall (total) yield is low (4%) on account of theinstability of the enzyme during the purification procedure mainly inthe stages of concentration by ultrafiltration when the concentration ofthe protein is low;

[0091] 2) the initial content of EH in the cellular extract ofAspergillus niger is low: a value of 0.4% of the soluble proteins iscalculated using the specific activity of the purified enzyme. However,the purified enzyme is responsible for all of the activity of the funguson pNSO. Thus, there is probably only one active protein on thissubstrate in Aspergillus niger.

[0092] The purified epoxide hydrolase (EH) has a single band in nativePAGE or SDS gel after staining with Coomassie Blue. Determinations ofthe activity of slices of gel obtained after electrophoresis of anon-denaturing polyacrylamide gel reveal a single band located at thesame level as the band of the labelled protein. The isoelectric point ofthe protein is 4.5 after determination by electrofocusing using a pHgradient from 3 to 9 and silver nitrate staining.

[0093] The EH of Aspergillus niger is a tetramer made up of fouridentical subunits of 45 kDa. The EHs from other sources are generallymonomeric or dimeric proteins. However, the epoxide hydrolase ofCorynebacterium sp. has recently been described as being dodecameric(Misawa et al., 1998).

[0094] The effect on the activity of several selective reagents wastested. EDTA and PMSF show no effect. Oxidizing agents such asmeta-chloroperbenzoic acid or hydrogen peroxide strongly inhibit theactivity of the enzyme. On the other hand, reducing agents such asβ-mercaptoethanol or cysteine show a positive effect on the enzyme'sactivity. Moreover, strong inactivation is observed with thiol blockingagents such as HgCl₂, 4-hydroxy-mercuribenzoate, iodoacetamide ordithionitrobenzene (DTNB). All these results demonstrate the essentialrole of one or more cysteine residue(s) on the activity of epoxidehydrolase. A similar effect is observed with the soluble EHs (sEH) frommammals (Wixtrom et al., 1985) and with the EH from Pseudomonas sp.(Rink et al., 1997), whereas the microsomal EHs (mEH) from mammals(Wixtrom et al., 1985) are not sensitive to thiol reagents.

[0095] The pH activity profile and the inhibition by ω-bromo-4-nitroacetophenone suggest the participation of a histidine residue in thecatalytic mechanism. Moreover, certain cysteine residues are importantfor the activity of the enzyme as demonstrated for mammalian sEH but notfor mEH (Wixtrom et al., 1985). The catalytic mechanism of mammalian sEHand mEH for the hydrolysis of epoxides has recently been elucidated(Beetham et al., 1995; Arand et al., 1996). A two-stage mechanisminvolving the formation of an intermediate covalent ester has beendemonstrated with the participation of two aspartic acids and onehistidine residue. However, little is known about the catalyticmechanism of microbial EHs. Recently, a similar mechanism wasdemonstrated for the epoxide hydrolase of the bacterium Agrobacteriumradiobacter (Rink et al., 1997). These elements suggest that the EH ofAspergillus niger uses a similar mechanism for the hydration of epoxidesas the mammalian EHs. This mechanism accords with the general process ofcatalysis demonstrated for the hydrolysis of para-substituted styreneoxide by a raw extract from Aspergillus niger (Pedragosa-Moreau et al.,1996).

[0096] With pNSO, addition of an organic solvent is required fordissolving the substrate. In fact, in the absence of co-solvent, noactivity can be detected. It was shown for other soluble EHs that theywere not active on micellar substrates (Hammock et al., 1997). Thus, theeffect of different co-solvents on the activity of epoxide hydrolasefrom Aspergillus niger was investigated. The nature of the co-solventshas a considerable influence on the yield in opening of the epoxide, thestrongest activities being obtained for DMF and acetone. The lowactivity obtained with THF could be correlated with inactivation of theenzyme by traces of peroxides that are usually present in the solvent.

[0097] The enzyme is active at a pH ranging from 5 to 9 with a maximumpeak at pH 7. The enzyme is active at a temperature ranging from 2 to45° C. with a maximum activity at 40° C. From 2 to 40° C. the activityincreases slightly (only 4 times) as indicated by the low activationenergy (27 kJ.mol⁻¹.° K⁻¹).

[0098] From the practical standpoint, the EH from Aspergillus niger isvery interesting for organic synthesis on account of its ability tohydrolyse racemic epoxides in a highly enantioselective manner. Theenantioselectivity is due to a higher affinity and a higher catalyticconstant for the (R) enantiomer of pNSO relative to the (S) enantiomer.

[0099] The ratio of the specific constant (k_(cat)/Km) shows that theinitial rate of hydrolysis of the (R) enantiomer is 55 times faster thanthat of the (S) enantiomer starting from racemic pNSO. This result issimilar to that obtained with whole cells on the same substrate(Pedragosa-Moreau, 1997). Moreover, the regioselectivity of the reactionis very high: 97% for the 2 carbon, as shown with the whole fungus(Pedragosa-Moreau, 1996). The enantioselectivity and theregioselectivity of hydrolysis of pNSO by the purified EH of Aspergillusniger are very similar to those determined with all of the cells.Accordingly, the purified enzyme is responsible for the entire activityof the fungus on pNSO.

B) Cloning and Characterization of the Soluble Epoxide Hyrolase fromAspergillus niger Which is Related to Mammalian Microsomal EpoxideHydrolases

[0100] I) Experimental Procedure

[0101] 1) Isolation of Nucleic Acids from Aspereillus niger (A. niger)

[0102]Aspergillus niger (the aforementioned strain No. LCP 521) wascultivated in a medium containing 10 g of glucose and 20 g of maizeliquor (Sigma, St. Louis, Cat. No. C4648) per litre of culture.Incubation was effected in a volume of 100 ml in a flask agitated at 28°C. for 3 days after inoculation with spores of the fungus. The myceliumis harvested by filtration on cloth and stored at −70° C. afterdetermination of the wet weight. Extraction of RNA is carried out by themethod of Chomczynski and Sacchi (1986) using 10 mL of denaturingsolution per gram of mycelium. The typical yield is 300 μg of total RNAper gram of mycelium. For isolation of the RNA, 2 g of mycelium ishomogenized with a Potter type of glass homogenizer in 15 mL of a lysissolution (solution of guanidine hydrochloride 6 M, containing 0.1 M ofsodium acetate, pH 5.5). After centrifugation at 10,000 g for 10 min,the supernatant is transferred to another tube and 2.5 volumes ofethanol are added. The precipitated nucleic acids are collected bycentrifugation at 10,000 g for 10 min and the resulting residue isdissolved overnight in 10 mL of lysis buffer after brief drying. Theinsoluble fraction is removed by centrifugation and the nucleic acidsare precipitated again by adding 25 mL of ethanol. The centrifugationpellet is washed with 70% ethanol, dried in air for 30 min and dissolvedin a TE buffer, pH 8.0.

[0103] 2) Cloning of the Gene of the EH of Aspergillus and of cDNA bythe Polymerase Chain Amplification Technique (Polymerase Chain Reaction,PCR)

[0104] The reverse PCR for amplification of the gene of the AspergillusEH was effected according to the following scheme: 500 ng of genomic DNAis digested with a suitable restriction enzyme (most of the successfulresults are obtained with BamHI or Cfol) and are recovered byprecipitation with ethanol after extraction with phenol/chloroformmixture. Of this 500 ng, 100 ng is circularized by ligation with DNAligase T4 (Life Technologies) in a volume of 20 μL in the conditionsspecified by the supplier. One microlitre of the resulting preparationwas amplified by PCR effected for 30 cycles (1 min 94° C., 1 min 60° C.,3 min 72° C.) with a DNA polymerase Taq (Perkin Elmer) in the standardreaction conditions recommended by the supplier. The primers used (MA2265′-ATGCGATCGGACTGCTGGACA-3′ and MA227 5′-CGCGGGCAATCCACACCTAC-3′)

[0105] are deducted from the sequence of a genomic fragment obtainedpreviously. An Xhol restriction site located between the two primingsites in the genomic sequence is used optionally for relinearizing thecircular DNA before the reverse PCR, in order to suppress the torsionalstress and so improve the efficiency of initial amplification of thegenomic support. The PCR products are separated by electrophoresis onagarose gel and the specific amplicons of the EH of Aspergillus areidentified by immunotransfer according to the Southern technique usingthe aforementioned genomic fragment as a probe. The fragments ofAspergillus EH gene identified in this way are purified byelectrophoresis on agarose gel using the Quiaex kit (Qiagen), and clonedin the pGEM-T vector (Promega) for sequence analyses by the chaintermination method.

[0106] On the basis of the information obtained from the sequence, 2primers

[0107] (MA290 5′-cggaattccATGgTCACTGGAGGAGCAATAATTAG-3′ and

[0108] MA291 5′-ttgaatTCCCTACTTCTGCCACAC-3′; the residues in capitalletters are complementary to the support sequence) surrounding theregion encoding the protein of the EH gene are deducted and used foramplifying the respective fragments of the genomic DNA and forreverse-transcribing the mRNA with high fidelity DNA polymerase Pfu(“Stratagene”) for 40 cycles (1 min 94° C., 1 min 50° C., 6 min 72° C.).The resulting DNA fragments are digested with EcoRI and inserted inpUC19 (New England Biolabs) for final sequence analysis.

[0109] 3) Expression, Purification and Analysis of Recombinant EpoxideHydrolase

[0110] For recombinant expression in E. coli, the cDNA fragment of theepoxide hydrolase of Aspergillus is amplified with a DNA polymerase Pfuusing the primer MA291 (see above) and the primer MA318

[0111] (5′gctgaattcacATGTCCGCTCCGTTCGCCAAG-3′)

[0112] in order to introduce an AfIIII Ncol-compatible recognition site(underlined in primer MA318) in the probable initiation codon of theepoxide hydrolase gene of Aspergillus which was revealed by sequenceanalysis.

[0113] The pGEF+ bacterial expression vector is modified by introducinga multiple cutting site

[0114] (5′-CCATGGGAATTCTCGAGATCTAAGCTTATGCATCAGCTGCATGG-3′)

[0115] in the Ncol site that contains the starting codon of the pGEF+vector in the context adapted to a ribosome binding site, upstream ofthe promoter of the RNA polymerase T7. The resulting plasmid is calledpGEF II hereinafter. The PCR fragment AfIII/Eco RI of the EH ofAspergillus is ligated in the Ncol/Eco RI site of pGEF II to produce thepGEF Asp EH″ expression construction. The E. coli strain BL21 (DE 3)(Novagen) is transformed with pGEF Asp EH and put in the LB medium at30° C. In late exponential phase, induction of expression of therecombinant protein is effected by adding isopropyl-β-thiogalactoside(100 μM). After two hours, the bacteria are collected by centrifugation,resuspended in 0.02 volumes of culture of the STE buffer (Tris-HCl, 10mM, sodium chloride 100 mM, ethylenediamine tetraacetic acid 1 mM, pH7.4) and stored at −70° C. Enzymatic activity is determined byconverting the R enantiomer of para-nitrostyrene oxide to thecorresponding diol. The reaction is carried out at a substrateconcentration of 880 μM in 500 μL STE at 37° C. for 30 min, in thepresence of 10 μL of acetonitrile which is used as solvent ofpara-nitrostyrene oxide.

[0116] The conversion reaction is terminated by extraction of thesubstrate with an equal volume of chloroform. In these conditions, morethan 99.9% of the substrate is extracted in the organic phase and 60% ofthe diol is recovered in the aqueous phase.

[0117] The conversion substrate is quantified by adding 400 μL ofsupernatant to 800 μL of water and reading the optical density at 277nM, with the molar extinction coefficient of the product being 9.1×10³M⁻¹ cm⁻¹. The epoxide hydrolase of Aspergillus is purified tohomogeneity by a three-stage procedure, according to the methoddescribed above.

[0118] Antibodies directed against the purified protein were obtained byimmunizing rabbits according to the technique described by Friedberg etal., 1991. The purified protein is analysed by SDS polyacrylamide gelelectrophoresis followed by labelling with Coomassie Blue or byimmunotransfer in accordance with the procedures published previously.

[0119] 4) Construction and Analysis of Epoxide Hydrolase Mutants

[0120] PCR-controlled, directed mutagenesis of the cDNA of epoxidehydrolase of Aspergillus is carried out by the method of Tomic et al.,1990, as described previously for soluble mammalian epoxide hydrolasesand microsomal epoxide hydrolases (Arand et al., 1996; Arand et al.,1999).

[0121] Primers were used for introducing various mutations. Themutations affecting the catalytic nucleophile Asp¹⁹² were introduced byreplacing the Ncol internal cassette of the cDNA of the epoxidehydrolase of Aspergillus with the PCR-modified fragment.

[0122] In addition, mutations targeting the residues of the charge relaysystem, namely Asp³⁴⁸ and His³⁷⁴, are introduced by replacing an XhoIfragment with the respective PCR fragment. The PCR modifications aregenerated using DNA polymerase Pfu so as to minimize the introduction ofunwanted sequence modifications. All the PCR-generated fragments arefinally sequenced to ensure they are correct. After recombinantexpression, the solubility of the mutant proteins is tested, whichrepresents an indicator of their structural integrity. After sonicationof the bacterial sediments, the resulting suspension is centrifuged at10,000 g, the pellet and the supernatant are tested for presence of theepoxide hydrolase of Aspergillus by immunotransfer. The enzymaticactivity is tested in the supernatant as described previously.

[0123] II) Results

[0124] Isolation of the gene of epoxide hydrolase (EH) of Aspergillusand the cDNA by reverse PCR, were obtained. It was difficult to obtainspecific amplified fragments using restriction enzymes with thehexameric recognition sites for digestion of the genomic DNA.

[0125] This seems to be due to two pairing errors in the MA226 primer,in comparison with the sequence of natural origin, which weakens theamplification of the long products, but poses no problem when usingrestriction enzymes with tetrameric recognition sequences. However, thefirst fragment obtained after restriction by BamHI of the DNA seems tobe artificially truncated, which is a consequence of the internalpriming of the initial amplicon. In consequence, the 3′ region of the EHof Aspergillus downstream from the genomic sequence is lacking in thisfragment and must be obtained separately in a second reverse PCRexperiment.

[0126] The epoxide hydrolase of Aspergillus is evidently related to themEHs of mammals, although this enzyme is unique in several respects.

[0127] First, it is a soluble enzyme that does not have an anchoringsequence in the membrane, in contrast to the microsomal epoxidehydrolases of mammals (mEHs), and their corresponding enzymes in thearthropods.

[0128] Second, the EH of Aspergillus has a much higher conversion powerwith para-nitrostyrene oxide, than that of mammalian epoxide hydrolaseswith their substrates.

[0129] Whereas the rat microsomal epoxide hydrolase (mEH) has a specificactivity with its model substrates styrene oxide and benzo[α]pyreneoxide of about 500 nmol converted per minute and milligram of pureenzyme, the epoxide hydrolase of Aspergillus hydrolyses 100 μmol of4-nitro-styrene oxide per minute and milligram of enzyme. The conversionnumber of rat mEH was increased by a factor of 30 by replacing the acidresidue of the charge relay system of its catalytic site, i.e. Glu⁴⁰⁴,with aspartic acid. Interestingly, the corresponding residue in thenative Aspergillus epoxide hydrolase is already an aspartic acid, incontrast to the fact that glutamic acid occupies this position in allthe other mEH enzymes. Substitution of catalytic Asp³⁴⁸ in the epoxidehydrolase of Aspergillus by Glu leads to a moderate decrease of Vmax bya factor of just 2. At the same time, the K_(M) fell by a factor of 3. Apossible explanation of this observation might be a reversal in thestage limiting the degree of conversion of the enzymatic reaction. Inthe mEH and sEH of mammals the second hydrolytic stage of the enzymaticreaction seems to be a stage limiting the degree of conversion. In suchconditions, i.e. when the rate constant of formation of the intermediateester k₁ is much greater than the constant k₂ for the hydrolytic stage,the decrease in Vmax due to a reduced k₂ occurs in parallel with asimilar decrease of K_(M), because K_(M)=K_(D)k₂/(k₁+k₂). However, if,initially k₁ limits the degree, and k₂ is much greater, the expressionk₂/(k₁+k₂) will be approximately equal to 1 and K_(M) is equal to K_(D).A halving of Vmax due to modulation of the charge relay system, i.e. ofthe important part of the catalytic site for the second stage of theenzymatic reaction, is probably due to a large decrease in k₂ of up tohalf the value of k₁. Consequently, k₂/(k₁+k₂) would now be close to ⅓,i.e. exactly the value observed for the Asp³⁴⁸ Glu mutant of the epoxidehydrolase (EH) of Aspergillus. Thus, these results are compatible withthe fact that in the case of EH of Aspergillus with para-nitrostyreneoxide as substrate, k₂ is greater than k₁, a situation that no longerexists in substitution of Asp³⁴⁸ by Glu. This would correspond exactlyto the scenario observed with the mEHs of mammals.

[0130] The structure of the gene of the epoxide hydrolase of Aspergillusis very complex, compared with the simplicity of the original organism.Whereas the average size of the introns identified is approx. 60 pb, andtherefore in agreement with that of many other genes of Aspergillus, incontrast the number of introns in the Aspergillus gene, 8 in all, isabnormally high.

[0131] None of the exons/introns is conserved between fungi and mammals,despite the identical number of introns in the 2 organisms. The fungusand mammal genes both have a first non-coding exon. In the rat, theexistence of at least 3 alternatives for the first exon has been noted.Here, the first non-coding exon permits the alternative use of differentpromoters for the synthesis of identical proteins.

C) EXAMPLES OF APPLICATION Example 1

[0132] 15 g of 1,1-diethoxybut-3-ene oxide (94 mmol or a concentrationof 0.3 mol per litre of reaction medium) is added to 300 ml of phosphatebuffer (pH 8, 0.1 M). The temperature is adjusted to 4° C. and 1.2 g ofpurified (native) enzyme is added. After stirring for 30 hours at 4° C.,the residual epoxide is extracted with pentane. Evaporation of thesolvent followed by distillation makes it possible to isolate 4.5 g of(S)-epoxide (yield=30%, ee=98%). Continuous extraction of the aqueousphase with dichloromethane makes it possible to isolate, afterpurification on a silica column, 9 g of (R)-diol (yield=54%, ee=47%).

Example 2

[0133] 6 g of para-bromo-α-methylstyrene oxide (28 mmol or aconcentration of 0.35 mol per litre of reaction medium) is added to 75ml of phosphate buffer (pH 8, 0.1 M). The temperature is adjusted to 4°C. and 0.35 g of purified (native) enzyme is added. After stirring for 8days at 0° C. the residual epoxide is extracted with pentane.Evaporation of the solvent followed by distillation makes it possible toisolate 2.3 g of (S)-epoxide (yield=39%, ee=99.7%). Continuousextraction of the aqueous phase with dichloromethane makes it possibleto isolate, after purification on a silica column, 3.19 g of (R)-diol(yield=49%, ee=96%).

Example 3

[0134] 4 g of para-chlorostyrene oxide (26 mmol or a concentration of 2mol per litre of reaction medium) is added to 9 ml of phosphate buffer(pH 7, 0.1 M). The temperature is adjusted to 0° C. and 2.3 g ofpurified (native) enzyme is added. After stirring for 8 hours at 0° C.the residual epoxide is extracted with pentane. Evaporation of thesolvent followed by distillation makes it possible to isolate 1.9 g of(S)-epoxide (yield=47%, ee=99%). Extraction of the aqueous phase withethyl acetate makes it possible to isolate, after purification on asilica column, 2.15 g of (R)-diol (yield=48%, ee=92%).

Example 4

[0135] 4 g of para-nitrostyrene oxide (24 mmol or a concentration of 0.3mol per litre of reaction medium) dissolved in 15 ml of DMSO is added to60 ml of phosphate buffer (pH 7, 0.1 M). The temperature is adjusted to27° C. and 0.7 g of purified (native) enzyme is added. After stirringfor 32 hours the reaction medium is saturated with NaCl then extractedcontinuously with dichloromethane. Evaporation of the solvent followedby chromatography on silica makes it possible to isolate 1.8 g of(S)-epoxide (yield=45%, ee=96%) and 2.3 g of (R)-diol (yield=52%,ee=86%).

Example 5

[0136] 1.5 g of para-isobutyl-α-methylstyrene oxide (7.9 mmol or aconcentration of 0.25 mol per litre of reaction medium) is added to 30ml of Tris buffer (pH 8, 0.4 M). The temperature is adjusted to 4° C.and 2.6 g of purified (native) enzyme is added. After stirring for 24days at 4° C. the residual epoxide is extracted with pentane.Evaporation of the solvent makes it possible to obtain the unpurified(S)-epoxide (ee=96%). Extraction of the aqueous phase with ether makesit possible to isolate, after purification on a silica column, 0.91 g of(R)-diol (yield=55%, ee=70%).

Example 6

[0137] 1 g of phenyl glycidyl ether (6.7 mmol or a concentration of 3.3mol per litre of reaction medium) is added to 1 ml of phosphate buffer(pH 7, 0.1 M). The temperature is adjusted to 27° C. and 25 mg ofpurified recombinant enzyme is added. After stirring for 15 hours at 27°C. all of the epoxide is converted to the corresponding racemic diol.Extraction with ethyl acetate makes it possible to isolate this diol ata quantitative yield.

BIBLIOGRAPHY

[0138] Audier H. E., Dupin J. F., Jullien J. (1968) Bull. Chem. Soc., 9,3844-3847.

[0139] Arand M., Wagner H., Oesch F. (1996) J. Biol. Chem., 271,4223-4229

[0140] Arand M., Müller F., Mecky A., Hinz W., Urban P., Pompon D.,Kellner R., Oesch F. (1999) Biochem. J., 337, 37-43.

[0141] Archelas A., Furstoss R. (1997) Ann. Rev. Microbiol., 51, 491-525

[0142] Archelas A., Furstoss R. (1998) Trends in Biotechnology, 16,108-116

[0143] Beetham J. K., Grant D., Arand M., Garbarino J., Kiyosue T.,Pinot F., Oesch F., Belknap W. R., shinozaki K., Hammock B. D. (1995)DNA Cell Biol., 14, 67-71

[0144] Blée E., Schuber F. (1992) Biochem. J., 282, 711-714

[0145] Borhan B., Jones A. D., Pinot F., Grant D. F., Kurth M. J.,Hammock B. D. (1995) Anal. Biochem., 231, 188-200

[0146] Chomczynski P., Sacchi N. (1986) Anal. Biochem., 162, 156-159

[0147] Dansette P. M., Makedonska V. B., Jerina D. M. (1978) Arch.Biochem. Biophys. 187, 290-298

[0148] Delaage M. (1968) Biochim. Biophys. Acta, 168, 443-445

[0149] Friedberg T., Kissel W., Arand M., Oesch F. (1991) In Methods inEnzymology (Waterman M. R. and Johnson E. F., eds) Vol. 206, pp.193-201, Academic Press, New York

[0150] Hammock B. D., Grant D. F., Storms D. H. (1997) In ComprehensiveToxicology (Sipes, I., McQueen C. and Gandolfi, A., Eds), pp 283-305,Pergamon Press, Oxford

[0151] Laemmli U. K. (1970) Nature, 227, 680-685

[0152] Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. (1951)J. Biol. Chem., 193, 265-275

[0153] Misawa E., Chan Kwo Chion C. K. C., Archer I. W., Woodland M. P.,Zhou N.Y., Carter S., Widdowson D. A., Leak D. A., Leak D. A. (1998)Eur. J. Biochem., 253, 173-183

[0154] Nellaiah H., Morisseau C., Archelas A., Furstoss R., Baratti J.C. (1996) Biotech. Bioeng., 49, 70-77

[0155] Pedragosa-Moreau S., Archelas A., Furstoss R. (1993) J. Org.Chem, 58, 5533-5536

[0156] Pedragosa-Moreau S., Archelas A., Furstoss R. (1995) Bull. Soc.Chem. Fr. 132, 769-800

[0157] Pedragosa-Moreau S., Archelas A., Furstoss R. (1996) J. Org.Chem. 61, 7402-7407

[0158] Pedragosa-Moreau S., Morisseau C., Zylber J., Baratti J. C.,Archelas A., Furstoss R. Tetrahedron(1997) 53, 9707-9714

[0159] Rink R., Fennema M., Smids M., Dehmel U., Janssen D. B. (1997) J.Biol. Chem., 272, 14650-14657

[0160] Schurig V., Betschinger F. (1992) Chem. Rev. 873-888

[0161] Tomic M., Sunjeravic I., Savtchenko E. S., Blumenberg M. (1990)Nucleic Acids Res., 18, 1656

[0162] Touhara K., Prestwitch G. D. (1993) J. Biol Chem., 268,19604-19609

[0163] Westkaemper R. B., Hanzlik R. P. (1980) Anal. Biochem., 102,63-67

[0164] Westkaemper R. B., Hanzlik R. P. (1981) Arch. Biochem. Biophys.,208, 195-204

[0165] Wixtrom R. N., Hammock B. D. (1985) In Biochemical Pharmacologyand Toxicology (Zakim D. and Vessey D. A., Eds) pp. 1-93, John Willey &Sons, New York.

1 8 1 1197 DNA Aspergillus niger CDS (1)..(1194) 1 atg tcc gct ccg ttcgcc aag ttt ccc tcg tcg gcg agc att tcg cct 48 Met Ser Ala Pro Phe AlaLys Phe Pro Ser Ser Ala Ser Ile Ser Pro 1 5 10 15 aat cct ttc acg gtctct atc ccg gat gaa cag ttg gat gac ttg aaa 96 Asn Pro Phe Thr Val SerIle Pro Asp Glu Gln Leu Asp Asp Leu Lys 20 25 30 acc ctc gtc cga ctg tccaag att gct cct ccc acc tat gag agc ctg 144 Thr Leu Val Arg Leu Ser LysIle Ala Pro Pro Thr Tyr Glu Ser Leu 35 40 45 caa gcg gat ggc cgg ttt ggcatc act tct gaa tgg ctg aca act atg 192 Gln Ala Asp Gly Arg Phe Gly IleThr Ser Glu Trp Leu Thr Thr Met 50 55 60 cgg gag aaa tgg ctc tcg gag tttgac tgg cga cca ttt gaa gct cga 240 Arg Glu Lys Trp Leu Ser Glu Phe AspTrp Arg Pro Phe Glu Ala Arg 65 70 75 80 ctg aac tct ttc cct cag ttt actaca gag atc gag ggt ctc acg att 288 Leu Asn Ser Phe Pro Gln Phe Thr ThrGlu Ile Glu Gly Leu Thr Ile 85 90 95 cac ttt gct gct ctc ttc tcc gag agggag gat gct gtg cct atc gca 336 His Phe Ala Ala Leu Phe Ser Glu Arg GluAsp Ala Val Pro Ile Ala 100 105 110 ttg ctc cat ggt tgg ccc ggc agc ttcgtt gag ttc tac cca atc ctg 384 Leu Leu His Gly Trp Pro Gly Ser Phe ValGlu Phe Tyr Pro Ile Leu 115 120 125 cag cta ttc cgg gag gag tac acc cctgag act ctg cca ttc cat ctg 432 Gln Leu Phe Arg Glu Glu Tyr Thr Pro GluThr Leu Pro Phe His Leu 130 135 140 gtt gtt ccg tcc ctt cct ggg tat actttt tca tct ggt ccc ccg ctg 480 Val Val Pro Ser Leu Pro Gly Tyr Thr PheSer Ser Gly Pro Pro Leu 145 150 155 160 gac aag gac ttc ggc ttg atg gacaac gcc cgg gtc gta gac cag ttg 528 Asp Lys Asp Phe Gly Leu Met Asp AsnAla Arg Val Val Asp Gln Leu 165 170 175 atg aag gac ctc ggg ttc gga agtggt tat att att cag gga ggt gat 576 Met Lys Asp Leu Gly Phe Gly Ser GlyTyr Ile Ile Gln Gly Gly Asp 180 185 190 att ggt agc ttt gtt gga cga ctgttg ggc gtg ggt ttc gac gcc tgc 624 Ile Gly Ser Phe Val Gly Arg Leu LeuGly Val Gly Phe Asp Ala Cys 195 200 205 aaa gcg gtt cat ttg aac ctg tgcgca atg agg gct ccc cct gag ggc 672 Lys Ala Val His Leu Asn Leu Cys AlaMet Arg Ala Pro Pro Glu Gly 210 215 220 ccg tca att gag agc ttg tcc gcagcg gag aag gag gga atc gcg cga 720 Pro Ser Ile Glu Ser Leu Ser Ala AlaGlu Lys Glu Gly Ile Ala Arg 225 230 235 240 atg gag aag ttc atg acc gatggc tta gct tat gcc atg gag cac agt 768 Met Glu Lys Phe Met Thr Asp GlyLeu Ala Tyr Ala Met Glu His Ser 245 250 255 act cgg ccc agt act att ggccac gtg ctg tcc agc agt ccg atc gca 816 Thr Arg Pro Ser Thr Ile Gly HisVal Leu Ser Ser Ser Pro Ile Ala 260 265 270 tta ctt gca tgg att ggt gagaaa tat ctc caa tgg gtg gat aaa ccc 864 Leu Leu Ala Trp Ile Gly Glu LysTyr Leu Gln Trp Val Asp Lys Pro 275 280 285 ctc cct tct gag acc atc ctcgag atg gtg agc ctg tat tgg ctg acg 912 Leu Pro Ser Glu Thr Ile Leu GluMet Val Ser Leu Tyr Trp Leu Thr 290 295 300 gaa agt ttc ccg cgg gca attcat acc tac cgc gag act acc cca act 960 Glu Ser Phe Pro Arg Ala Ile HisThr Tyr Arg Glu Thr Thr Pro Thr 305 310 315 320 gcc tcc gct ccc aat ggagcg aca atg ctt cag aag gaa tta tat att 1008 Ala Ser Ala Pro Asn Gly AlaThr Met Leu Gln Lys Glu Leu Tyr Ile 325 330 335 cac aag ccg ttt ggg ttctcc ttc ttc ccc aag gac ctt tgt cct gtg 1056 His Lys Pro Phe Gly Phe SerPhe Phe Pro Lys Asp Leu Cys Pro Val 340 345 350 cct cgg agc tgg att gctaca acg gga aat cta gta ttc ttc cgg gat 1104 Pro Arg Ser Trp Ile Ala ThrThr Gly Asn Leu Val Phe Phe Arg Asp 355 360 365 cat gca gag gga gga cacttt gcc gca ttg gag cgt cca cgc gag ctg 1152 His Ala Glu Gly Gly His PheAla Ala Leu Glu Arg Pro Arg Glu Leu 370 375 380 aag acc gac ctg aca gcattt gtc gag cag gtg tgg cag aag tag 1197 Lys Thr Asp Leu Thr Ala Phe ValGlu Gln Val Trp Gln Lys 385 390 395 2 398 PRT Aspergillus niger 2 MetSer Ala Pro Phe Ala Lys Phe Pro Ser Ser Ala Ser Ile Ser Pro 1 5 10 15Asn Pro Phe Thr Val Ser Ile Pro Asp Glu Gln Leu Asp Asp Leu Lys 20 25 30Thr Leu Val Arg Leu Ser Lys Ile Ala Pro Pro Thr Tyr Glu Ser Leu 35 40 45Gln Ala Asp Gly Arg Phe Gly Ile Thr Ser Glu Trp Leu Thr Thr Met 50 55 60Arg Glu Lys Trp Leu Ser Glu Phe Asp Trp Arg Pro Phe Glu Ala Arg 65 70 7580 Leu Asn Ser Phe Pro Gln Phe Thr Thr Glu Ile Glu Gly Leu Thr Ile 85 9095 His Phe Ala Ala Leu Phe Ser Glu Arg Glu Asp Ala Val Pro Ile Ala 100105 110 Leu Leu His Gly Trp Pro Gly Ser Phe Val Glu Phe Tyr Pro Ile Leu115 120 125 Gln Leu Phe Arg Glu Glu Tyr Thr Pro Glu Thr Leu Pro Phe HisLeu 130 135 140 Val Val Pro Ser Leu Pro Gly Tyr Thr Phe Ser Ser Gly ProPro Leu 145 150 155 160 Asp Lys Asp Phe Gly Leu Met Asp Asn Ala Arg ValVal Asp Gln Leu 165 170 175 Met Lys Asp Leu Gly Phe Gly Ser Gly Tyr IleIle Gln Gly Gly Asp 180 185 190 Ile Gly Ser Phe Val Gly Arg Leu Leu GlyVal Gly Phe Asp Ala Cys 195 200 205 Lys Ala Val His Leu Asn Leu Cys AlaMet Arg Ala Pro Pro Glu Gly 210 215 220 Pro Ser Ile Glu Ser Leu Ser AlaAla Glu Lys Glu Gly Ile Ala Arg 225 230 235 240 Met Glu Lys Phe Met ThrAsp Gly Leu Ala Tyr Ala Met Glu His Ser 245 250 255 Thr Arg Pro Ser ThrIle Gly His Val Leu Ser Ser Ser Pro Ile Ala 260 265 270 Leu Leu Ala TrpIle Gly Glu Lys Tyr Leu Gln Trp Val Asp Lys Pro 275 280 285 Leu Pro SerGlu Thr Ile Leu Glu Met Val Ser Leu Tyr Trp Leu Thr 290 295 300 Glu SerPhe Pro Arg Ala Ile His Thr Tyr Arg Glu Thr Thr Pro Thr 305 310 315 320Ala Ser Ala Pro Asn Gly Ala Thr Met Leu Gln Lys Glu Leu Tyr Ile 325 330335 His Lys Pro Phe Gly Phe Ser Phe Phe Pro Lys Asp Leu Cys Pro Val 340345 350 Pro Arg Ser Trp Ile Ala Thr Thr Gly Asn Leu Val Phe Phe Arg Asp355 360 365 His Ala Glu Gly Gly His Phe Ala Ala Leu Glu Arg Pro Arg GluLeu 370 375 380 Lys Thr Asp Leu Thr Ala Phe Val Glu Gln Val Trp Gln Lys385 390 395 3 21 DNA Artificial Sequence Description of ArtificialSequence Primer 3 atgcgatcgg actgctggac a 21 4 20 DNA ArtificialSequence Description of Artificial Sequence Primer 4 cgcgggcaatccacacctac 20 5 35 DNA Artificial Sequence Description of ArtificialSequence Primer 5 cggaattcca tggtcactgg aggagcaata attag 35 6 24 DNAArtificial Sequence Description of Artificial Sequence Primer 6ttgaattccc tacttctgcc acac 24 7 32 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 7 gctgaattca catgtccgct ccgttcgcca ag 32 844 DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 8 ccatgggaat tctcgagatc taagcttatg catcagctgc atgg 44

1. A protein of fungal origin having epoxide hydrolase activity, such asis obtained in essentially pure form by extraction from cells of fungi,or by culture of host cells transformed by a nucleotide sequence codingfor the aforementioned fungal protein, or protein derived bysubstitution, suppression or addition of one or more amino acids of theaforementioned protein of fungal origin and possessing epoxide hydrolaseactivity.
 2. A protein according to claim 1, characterized in that itcomprises: the sequence SEQ ID NO: 2, or any sequence derived from thesequence SEQ ID NO: 2, especially by substitution, suppression oraddition of one or more amino acids, and possessing epoxide hydrolaseactivity, the said derived sequence preferably having a homology of atleast about 40% with the sequence SEQ ID NO: 2, or any fragment of thesequence SEQ ID NO: 2, or of a sequence derived from the latter asdefined above, and possessing epoxide hydrolase activity, the saidfragment preferably consisting of at least about 10 amino acids that arecontiguous in the region delimited by the amino acids located inpositions 1 and 339 of the sequence SEQ ID NO:
 2. 3. A protein accordingto claim 1 or 2, characterized in that it corresponds to a fungalepoxide hydrolase in essentially pure form, such as is obtained byextraction and purification from cultures of cells of fungi of theAspergillus species.
 4. A protein according to one of the claims 1 to 3,characterized in that it corresponds to the fungal epoxide hydrolase inessentially pure form represented by SEQ ID NO: 2, such as is obtainedby extraction and purification from cultures of cells of strains ofAspergillus niger or of Aspergillus turingensis.
 5. A protein accordingto claim 1 or 2, characterized in that it corresponds to a recombinantfungal epoxide hydrolase, such as is obtained in essentially pure formby transformation of suitable host cells by means of vectors containing:the nucleotide sequence SEQ ID NO: 1 encoding the epoxide hydrolaserepresented by SEQ ID NO: 2, or any sequence derived from SEQ ID NO: 1by degeneration of the genetic code, and encoding the epoxide hydrolaserepresented by SEQ ID NO: 2, or any sequence derived from the sequenceSEQ ID NO: 1, especially by substitution, suppression or addition of oneor more nucleotides, and coding for an enzyme possessing epoxidehydrolase activity, the said derived sequence preferably having ahomology of at least about 45% with the sequence SEQ ID NO: 1, or anyfragment of the sequence SEQ ID NO: 1, or of a sequence derived from thelatter as defined above, and coding for an enzyme possessing epoxidehydrolase activity, the said fragment preferably consisting of at leastabout 20 nucleotides that are contiguous in the region delimited by thenucleotides located in positions 1 and 1197 of the sequence SEQ IDNO:
 1. 6. A protein according to claim 5, characterized in that itcorresponds to the fungal recombinant epoxide hydrolase represented bySEQ ID NO: 2, such as is obtained by transformation of suitable hostcells by means of vectors containing the nucleotide sequence SEQ ID NO:1, or any sequence derived from SEQ ID NO: 1 by degeneration of thegenetic code, and encoding the epoxide hydrolase represented by SEQ IDNO:
 2. 7. A nucleotide sequence encoding a protein of fungal origin withepoxide hydrolase activity such as is defined by one of the claims 1 to6.
 8. A nucleotide sequence according to claim 7, characterized in thatit comprises: the sequence represented by SEQ ID NO: 1 encoding theepoxide hydrolase represented by SEQ ID NO: 2, or any sequence derivedfrom the sequence SEQ ID NO: 1 by degeneration of the genetic code, andencoding the epoxide hydrolase represented by SEQ ID NO: 2, or anysequence derived from the sequence SEQ ID NO: 1, especially bysubstitution, suppression or addition of one or more nucleotides, andcoding for an enzyme possessing epoxide hydrolase activity, the saidderived sequence preferably having a homology of at least about 45% withthe sequence SEQ ID NO: 1, or any fragment of the sequence SEQ ID NO: 1,or of a sequence derived from the latter as defined above, and codingfor an enzyme possessing epoxide hydrolase activity, the said fragmentpreferably consisting of at least about 20 nucleotides that arecontiguous in the region delimited by the nucleotides located inpositions 1 and 1197 of the sequence SEQ ID NO: 1, or any complementarynucleotide sequence of the aforementioned sequences or fragments, or anynucleotide sequence coding for an enzyme possessing epoxide hydrolaseactivity, and capable of hybridization with one of the aforementionedsequences or fragments, the aforementioned sequences or fragments beingof single-stranded or double-stranded form.
 9. A vector, especially aplasmid, containing a nucleotide sequence according to claim 7 or
 8. 10.A host cell, in particular chosen from bacteria, viruses, yeasts, fungi,plants or mammalian cells, the said host cell being transformed,especially by means of a vector according to claim 9, in such a way thatits genome contains a nucleotide sequence according to claim 7 or
 8. 11.The use of proteins with epoxide hydrolase activity defined in one ofthe claims 1 to 6, as enzymatic biocatalysts in the implementation ofmethods of preparation of epoxides or of enantiomerically pure vicinaldiols, especially in the pharmaceutical and plant-protection field, orin the field of manufacture of specific optical materials.
 12. A methodof preparation of epoxides and/or of enantiomerically pure diolsrespectively of the following formulae (II) and (III)

in which R₁, R₂, R₃ and R₄ represent any groups, especially groups thatare characteristic of pharmaceutical and plant-protection compounds, orof specific optical materials corresponding to the said epoxides orvicinal diols, the said method comprising a stage of treatment of amixture of diastereoisomeric epoxides, or of a chiral epoxide in racemicform, or of a prochiral epoxide of the following formula (I):

with a protein with epoxide hydrolase activity according to one of theclaims 1 to 6, or with the host cells according to claim 10 expressing aprotein with epoxide hydrolase activity according to one of the claims 1to 6, which leads to the production of: a mixture of the aforementionedcompounds of formulae (II) and (III), it being possible, if necessary,for the said compounds of formulae (II) and (III) to be separated by anadditional stage of purification, or of just the aforementioned compoundof formula (III).
 13. A method of preparation of a protein withrecombinant epoxide hydrolase activity according to claim 5 or 6,characterized in that it comprises a stage of transformation of hostcells, preferably chosen from the bacteria, viruses, yeasts, fungi,plants or mammalian cells, with a vector according to claim 9, and astage of purification of the recombinant epoxide hydrolase produced bythe said cells.
 14. A method of preparation of a protein with epoxidehydrolase activity in essentially pure form according to claim 3 or 4,the said method comprising: a stage of extraction of the enzyme fromcellular cultures of fungi, such as fungi of the Aspergillus species,especially by crushing the fungus using a press, followed by a stage oflow-speed centrifugation, recovery of the supernatant, and, if required,concentration, a stage of purification of the enzyme from the extractobtained in the preceding stage, especially by successive passagesthrough columns of DEAE-Sepharose, Phenyl-Sepharose, Mono Q and Superose12.